Early treatment of HER2-amplified brain tumors with targeted NK-92 cells and focused ultrasound improves survival

Abstract

Background: Malignant brain tumors have a dismal prognosis, with residual tumor remaining after surgery necessitating adjuvant chemoradiotherapy. The blood-brain barrier hinders many chemotherapeutic agents, resulting in modest treatment efficacy. We previously demonstrated that targeted natural killer (NK)-92 cells could be delivered to desired regions of the brain using MRI-guided focused ultrasound and Definity microbubbles. Targeted NK-92 cells have advantages over many systemic therapies including their specific cytotoxicity to malignant cells (particularly those expressing the target antigen), ability to spare healthy cells, and being unaffected by efflux channels.

Methods: We investigated whether longitudinal treatments with targeted NK-92 cells, focused ultrasound, and microbubbles could slow tumor growth and improve survival in an orthotopic HER2-amplified rodent brain tumor model using a human breast cancer line as a prototype. The HER2 receptor, involved in cell growth and differentiation, is expressed by both primary and metastatic brain tumors. Breast cancers with HER2 amplification have a higher risk of CNS metastasis and poorer prognosis.

Results: Early intensive treatment with targeted NK-92 cells and ultrasound improved survival compared with biweekly treatments or either treatment alone. The intensive treatment paradigm resulted in long-term survival in 50% of subjects.

Conclusions: Many tumor proteins could be exploited for targeted therapy with the NK-92 cell line; combined with the mounting safety evidence for transcranial ultrasound, these results may soon be translatable to a highly targeted treatment option for patients with brain tumors.

Keywords: MRIgFUS; breast cancer; focused ultrasound; immune cell therapy.

Fig. 1.

A schematic representation of the treatment setup, effect, and organization of the study. (A) The focused ultrasound (FUS) treatment setup, with the focused transducer (551.5 kHz) coupled to the rodent via a bath of degassed water and targeted using MRI. (B) NK-92 cells extravasate from the cerebrovasculature following interaction with FUS and microbubbles at the site of the tumor 11. (C) NK-92 cells labeled with iron and stained with Prussian blue following FUS. The cells are tracking toward the tumor, denoted by the star (★). (D) The 2 treatment arms, with the diamonds each representing a treatment event. In each arm, animals were randomized to receive FUS+Cells, FUS, or Cells for the duration of the study.

Fig. 2.

Representative images of blood-brain barrier disruption (BBBD). The contrast enhancement in the tumor before (A) and after BBBD (B and C) are shown. The focus is larger than the tumor size, so increased permeability is seen in the peritumoral area, favoring the medial aspect of the tumor due to the curvature of the rat skull. The coronal image (C) shows heterogeneity in the enhancement pattern due to standing waves. There was no difference in the enhancement after BBBD between the focused ultrasound (FUS) and FUS+Cells groups in either arm.

Fig. 3.

Comparison of the distributed and front-loaded treatment arms. A and E are representative contrast-enhanced T1 MR images of the tumor progression at 14 days. (B–D) The tumor volumes, animal weights, and survival data for the distributed arm. (F–H) Same data for front-loaded arm. F and H show significant reduction in tumor volume and increase in survival, respectively, achieved with an aggressive treatment schedule. (G) The front-loaded schedule was taxing on the animals. An initial weight loss was measured in the animals, whereas this was not observed in the distributed arm. Statistically significant results are denoted by *.

Fig. 4.

Representative T2-weighted 7T MR images of the long-term survivors in the focused ultrasound (FUS) + Cells group of the front-loaded arm. The tumor location is indicated by the hashed circle. There is some signal heterogeneity in the frontal striatum and some tissue loss suggested by the increased prominence of the right frontal horn of the lateral ventricle. There were no blood products seen on postmortem examination to explain the MR signal. It is impossible to distinguish whether the atrophy was due to the treatment or the tumor. The long-term survivors were completely asymptomatic until the end of the study.

Fig. 5.

Histopathological results depicting tumor progression and cell populations in the front-loaded arm. A–C depict H&E stained sections in the focused ultrasound (FUS) + Cells group (A) and (B) and the Cell group (C). The corresponding higher magnification images of HER2 expression are shown in D–F. The cystic (★) and necrotic (×) regions are identified in A and C, preventing NK-92 cells from circulating through and accumulating in parts of the tumor. There are virtually no HER2-positive cells in D, suggesting that this subpopulation was wiped out by the targeted NK-92 cells. Furthermore, in E, the leptomeningeal disease and the portion of the tumor closest to the skull were more uniformly HER2-positive; these areas were somewhat sheltered from the FUS treatment due to lensing effects of the rodent skull. The tumor farthest from the skull, more readily targeted by the FUS, is again void of HER2-positive cells. There was some variability of HER2 expression even in the Cell group (F), where we didn't expect the targeted NK-92 cells to have any effect. Representative H&E of one of the long-term survivors (G), with a high-magnification view of the tumor region in H (field of view 88 μm). The ventricular prominence can again be seen (G) without any evidence of hemorrhage or residual tumor (G, H). Representative CD-45 staining, showing no evidence of NK-92 cells remaining at the time of sacrifice (I).